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. 1995 Feb;69(2):1044–1049. doi: 10.1128/jvi.69.2.1044-1049.1995

Intracellular interference of tick-borne flavivirus infection by using a single-chain antibody fragment delivered by recombinant Sindbis virus.

W Jiang 1, K Venugopal 1, E A Gould 1
PMCID: PMC188675  PMID: 7815482

Abstract

A single-chain antibody fragment that identifies a neutralizing epitope on the envelope protein of louping ill and some other tick-borne flaviviruses was previously expressed in soluble form from bacteria and shown to be functionally active in vitro. To see whether or not the single-chain antibody could bind and inactivate infectious virus in vivo, we have used recombinant Sindbis virus as a delivery vehicle for intracellular expression of the antibody fragment. The variable genes and interchain linker encoding the single-chain antibody were cloned into a double subgenomic Sindbis virus expression vector to generate recombinant Sindbis virus. Infection with this recombinant Sindbis virus provided high-level cytoplasmic expression of the antibody fragment in mammalian cells. We demonstrate (i) that the antibody fragment was antigen binding and (ii) that louping ill virus infectivity was significantly reduced in the presence of intracellular antibody expressed by the superinfecting recombinant Sindbis virus.

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Selected References

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  1. Biocca S., Neuberger M. S., Cattaneo A. Expression and targeting of intracellular antibodies in mammalian cells. EMBO J. 1990 Jan;9(1):101–108. doi: 10.1002/j.1460-2075.1990.tb08085.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Biocca S., Pierandrei-Amaldi P., Campioni N., Cattaneo A. Intracellular immunization with cytosolic recombinant antibodies. Biotechnology (N Y) 1994 Apr;12(4):396–399. doi: 10.1038/nbt0494-396. [DOI] [PubMed] [Google Scholar]
  3. Boulton R. W., Westaway E. G. Replication of the flavivirus Kunjin: proteins, glycoproteins, and maturation associated with cell membranes. Virology. 1976 Feb;69(2):416–430. doi: 10.1016/0042-6822(76)90473-6. [DOI] [PubMed] [Google Scholar]
  4. Bredenbeek P. J., Frolov I., Rice C. M., Schlesinger S. Sindbis virus expression vectors: packaging of RNA replicons by using defective helper RNAs. J Virol. 1993 Nov;67(11):6439–6446. doi: 10.1128/jvi.67.11.6439-6446.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carlson J. R. A new means of inducibly inactivating a cellular protein. Mol Cell Biol. 1988 Jun;8(6):2638–2646. doi: 10.1128/mcb.8.6.2638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carlson J. R. A new use for intracellular antibody expression: inactivation of human immunodeficiency virus type 1. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7427–7428. doi: 10.1073/pnas.90.16.7427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chanas A. C., Gould E. A., Clegg J. C., Varma M. G. Monoclonal antibodies to Sindbis virus glycoprotein E1 can neutralize, enhance infectivity, and independently inhibit haemagglutination or haemolysis. J Gen Virol. 1982 Jan;58(Pt 1):37–46. doi: 10.1099/0022-1317-58-1-37. [DOI] [PubMed] [Google Scholar]
  8. Chen S. Y., Khouri Y., Bagley J., Marasco W. A. Combined intra- and extracellular immunization against human immunodeficiency virus type 1 infection with a human anti-gp120 antibody. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):5932–5936. doi: 10.1073/pnas.91.13.5932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Duan L., Bagasra O., Laughlin M. A., Oakes J. W., Pomerantz R. J. Potent inhibition of human immunodeficiency virus type 1 replication by an intracellular anti-Rev single-chain antibody. Proc Natl Acad Sci U S A. 1994 May 24;91(11):5075–5079. doi: 10.1073/pnas.91.11.5075. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  10. Dul J. L., Argon Y. A single amino acid substitution in the variable region of the light chain specifically blocks immunoglobulin secretion. Proc Natl Acad Sci U S A. 1990 Oct;87(20):8135–8139. doi: 10.1073/pnas.87.20.8135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fuerst T. R., Niles E. G., Studier F. W., Moss B. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8122–8126. doi: 10.1073/pnas.83.21.8122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hahn C. S., Hahn Y. S., Braciale T. J., Rice C. M. Infectious Sindbis virus transient expression vectors for studying antigen processing and presentation. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2679–2683. doi: 10.1073/pnas.89.7.2679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Higgs S., Powers A. M., Olson K. E. Alphavirus expression systems: applications to mosquito vector studies. Parasitol Today. 1993 Dec;9(12):444–452. doi: 10.1016/0169-4758(93)90098-z. [DOI] [PubMed] [Google Scholar]
  14. Jiang W. R., Lowe A., Higgs S., Reid H., Gould E. A. Single amino acid codon changes detected in louping ill virus antibody-resistant mutants with reduced neurovirulence. J Gen Virol. 1993 May;74(Pt 5):931–935. doi: 10.1099/0022-1317-74-5-931. [DOI] [PubMed] [Google Scholar]
  15. Jiang W., Bonnert T. P., Venugopal K., Gould E. A. A single chain antibody fragment expressed in bacteria neutralizes tick-borne flaviviruses. Virology. 1994 Apr;200(1):21–28. doi: 10.1006/viro.1994.1158. [DOI] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Liljeström P., Garoff H. A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Biotechnology (N Y) 1991 Dec;9(12):1356–1361. doi: 10.1038/nbt1291-1356. [DOI] [PubMed] [Google Scholar]
  18. Marasco W. A., Haseltine W. A., Chen S. Y. Design, intracellular expression, and activity of a human anti-human immunodeficiency virus type 1 gp120 single-chain antibody. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7889–7893. doi: 10.1073/pnas.90.16.7889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Morgan D. O., Roth R. A. Analysis of intracellular protein function by antibody injection. Immunol Today. 1988 Mar;9(3):84–88. doi: 10.1016/0167-5699(88)91270-4. [DOI] [PubMed] [Google Scholar]
  20. Munro S., Pelham H. R. An Hsp70-like protein in the ER: identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein. Cell. 1986 Jul 18;46(2):291–300. doi: 10.1016/0092-8674(86)90746-4. [DOI] [PubMed] [Google Scholar]
  21. Ng M. L., Pedersen J. S., Toh B. H., Westaway E. G. Immunofluorescent sites in vero cells infected with the flavivirus Kunjin. Arch Virol. 1983;78(3-4):177–190. doi: 10.1007/BF01311313. [DOI] [PubMed] [Google Scholar]
  22. Powers A. M., Olson K. E., Higgs S., Carlson J. O., Beaty B. J. Intracellular immunization of mosquito cells to LaCrosse virus using a recombinant Sindbis virus vector. Virus Res. 1994 Apr;32(1):57–67. doi: 10.1016/0168-1702(94)90061-2. [DOI] [PubMed] [Google Scholar]
  23. Rice C. M., Levis R., Strauss J. H., Huang H. V. Production of infectious RNA transcripts from Sindbis virus cDNA clones: mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants. J Virol. 1987 Dec;61(12):3809–3819. doi: 10.1128/jvi.61.12.3809-3819.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Shiu S. Y., Ayres M. D., Gould E. A. Genomic sequence of the structural proteins of louping ill virus: comparative analysis with tick-borne encephalitis virus. Virology. 1991 Jan;180(1):411–415. doi: 10.1016/0042-6822(91)90048-g. [DOI] [PubMed] [Google Scholar]
  25. Tavladoraki P., Benvenuto E., Trinca S., De Martinis D., Cattaneo A., Galeffi P. Transgenic plants expressing a functional single-chain Fv antibody are specifically protected from virus attack. Nature. 1993 Dec 2;366(6454):469–472. doi: 10.1038/366469a0. [DOI] [PubMed] [Google Scholar]
  26. Venugopal K., Buckley A., Reid H. W., Gould E. A. Nucleotide sequence of the envelope glycoprotein of Negishi virus shows very close homology to louping ill virus. Virology. 1992 Sep;190(1):515–521. doi: 10.1016/0042-6822(92)91245-p. [DOI] [PubMed] [Google Scholar]
  27. Ward E. S., Güssow D., Griffiths A. D., Jones P. T., Winter G. Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature. 1989 Oct 12;341(6242):544–546. doi: 10.1038/341544a0. [DOI] [PubMed] [Google Scholar]
  28. Westaway E. G., Ng M. L. Replication of flaviviruses: separation of membrane translation sites of Kunjin virus proteins and of cell proteins. Virology. 1980 Oct 15;106(1):107–122. doi: 10.1016/0042-6822(80)90226-3. [DOI] [PubMed] [Google Scholar]
  29. Winter G., Milstein C. Man-made antibodies. Nature. 1991 Jan 24;349(6307):293–299. doi: 10.1038/349293a0. [DOI] [PubMed] [Google Scholar]
  30. Xiong C., Levis R., Shen P., Schlesinger S., Rice C. M., Huang H. V. Sindbis virus: an efficient, broad host range vector for gene expression in animal cells. Science. 1989 Mar 3;243(4895):1188–1191. doi: 10.1126/science.2922607. [DOI] [PubMed] [Google Scholar]

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